Transglutaminases are Ca.sup.2+ dependent enzymes that catalyze the formation of isopeptide bonds in proteins between the side chain .gamma.-carboxamide group of glutamine and the side chain .epsilon.-amino group of lysine. Transglutaminases are found both extracellularly and intracellularly. Examples of transglutaminases include Factor XIII, epidermal transglutaminases, type II transglutaminases (tissue type transglutaminase, liver transglutaminases), and type I transglutaminases (keratinocyte transglutaminase).
Factor XIII (F XIII, fibrin-stabilizing factor), is a transglutaminase found as a proenzyme in plasma, platelets and monocytes/macrophages. It is important, inter alia, for ensuring efficient blood coagulation and would healing. F XIII, isolated from placenta or plasma or in the form of fresh frozen plasma, has ben employed for many years for treatment of factor XIII deficiency. It recently has become possible to prepare factor XIII using recombinant DNA technology. As used herein, rF XIII refers to recombinant factor XIII.
Commercially available purified or partially purified transglutaminase or F XIII preparations contain added stabilizers, such as human serum albumin (HSA). The use of protein stabilizers is problematic, since it decreases protein specific activity, increases the protein load when administered to patients, and may interfere with assessment of purity. It may also make the protein preparation immunogenic. These disadvantages of protein stabilizers make them particularly disadvantageous for stabilizing highly purified proteins, such as recombinant proteins. Additionally, use of protein stabilizers causes potential contamination with viral antigens when albumin, for example, is added.
The composition and activity of protein preparations used in therapy must be stable over relatively long periods of time. It is only rarely possible to achieve this stability in solution and, therefore, such products are frequently marketed in the dry state. Mild freeze-drying (lyophilization) is the method of choice for drying such products. However, even when this method is used, stable preparations fulfilling the requirements for integrity and durability are difficult to achieve.
Freeze-drying of unstabilized transglutaminase solutions leads, for example, to a marked drop in activity and to considerable turbidity. Formulations based on albumin and containing relatively high concentrations of salts have, therefore, been previously described for use with purified F XIII preparations. See, for example, DE-C-2063 070 and JP 53/59018. These formulations, however, suffer from the disadvantage described above of containing foreign protein, with all the problems attached thereto.
The freeze-drying of rF XIII in the presence of glycine or arginine and non-reducing sugars has been described. See WO 90/03147. Neither the stability, solubility, nor clarity of the reconstituted lyophilisate was described, however. The products obtained were stored at -20.degree. C., indicating that the lyophilized material probably had inadequate stability at 4.degree. C.
It is apparent, therefore, that a stable lyophilized formulation for transglutaminases, in particular for F XIII, is greatly to be desired. It is desirable such a formulation can be administered locally (e.g. topically) or parenterally, is stable at 2-8.degree. C. (or higher) and does not require the addition of protein stabilizers, for example, HSA. Furthermore, the lyophilisate should be readily soluble and, following dissolution, should yield a stable, non-turbid solution.